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Identification and Characterization of the Forkhead Box

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Identification and Characterization of the Forkhead Box IDENTIFICATION AND CHARACTERIZATION OF THE FORKHEAD BOX FAMILY OF TRANSCRIPTIONAL REGULATORS IN PARASITIC SCHISTOSOMES by MELISSA M. VARRECCHIA Submitted in partial fulfillment of the requirements for the degree of Doctor of philosophy Department of Biology CASE WESTERN RESERVE UNIVERSITY August 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Melissa M. Varrecchia candidate for the degree of Doctor of Philosophy Committee Chair Michael F. Benard Committee Member Emmitt R. Jolly Committee Member Christopher A. Cullis Committee Member Claudia M. Mizutani Committee Member Brian M. McDermott Date of Defense June 6, 2017 *We also certify that written approval has been obtained for any proprietary material contained therein. ii Dedication I would like to dedicate this dissertation to my Mom and Dad. Mom, thank you for your endless love, support and encouragement throughout the years. Dad, I miss you and I know that you are with me always, cheering me on in spirit. iii Table of Contents Table of Contents………………………………………………………………………...1 List of Tables……………………………………………………………………………..6 List of Figures…………………………………………………………………………....8 Acknowledgements…………………………………………………………………..…11 List of Abbreviations…………………………………………………………………...13 Abstract…………………………………………………………………………………15 Chapter 1: Introduction………………………………………………………………..17 1.1 Schistosomiasis………………………………………………………………17 1.2 Pathogenesis and treatment…………………………………………………..18 1.3 Schistosome life cycle………………………………………………………..20 1.4 Schistosome morphology and development…………………………………21 1.5 Forkhead box transcription factors…………………………………………..23 1.6 Fox family subclasses and development………………………………….….25 1.7 Forkhead box genes in parasites……………………………………………..26 1.8 Thesis aims and significance…………………………………………….......29 Chapter 2: Schistosome Fox genes: SmFoxA-SmFoxG……………………………...31 2.1 Materials and methods……………………………………………………………..31 1 2.1.1 Animals and parasites……………………………………………………...31 2.1.2 Bioinformatics……………………………………………………………...31 2.1.3 Cloning and sequencing………………………………………………........33 2.1.4 Yeast transformation and modified yeast one-hybrid……………………...34 2.1.5 Absolute quantitative PCR……………………………………………........36 2.2 Results……………………………………………………………………………….37 2.2.1 Schistosomes have forkhead genes from several subclasses of the Fox family of transcription factors …………………………………………………...37 2.2.2 Schistosome forkhead transcripts are developmentally regulated………....45 2.2.3 Schistosome forkhead proteins are transcriptional regulators……………..48 2.3 Supplementary information………………………………………………………..53 2.3.1 Supplementary figures……………………………………………………..53 2.3.2 Supplementary tables………………………………………………………55 Chapter 3: Schistosome Fox genes: SmFoxJ-SmFoxP……………………………….60 3.3 Materials and methods……………………………………………………………..60 3.1.1 Animals and parasites……………………………………………………...60 3.1.2 Bioinformatics…………………………………………………….………..60 3.1.3 Cloning and Sequencing…………………………………………………...61 2 3.1.4 Yeast Transformation and Modified Yeast One-hybrid…………………...63 3.1.5 Absolute quantitative PCR…………………………………………………64 3.2 Results……………………………………………………………………………….65 3.2.1 Several subclasses of the Fox family of transcriptional regulators were identified in schistosome worms…………………………………………………65 3.2.2 Developmental regulation of schistosome forkhead transcript expression………………………………………………………………………..72 3.2.3 Schistosome forkhead proteins regulate transcription……………………..75 3.3 Supplementary Information…………………………………………………….....82 3.3.1 Supplementary figures……………………………………………………..82 3.3.2 Supplementary tables………………………………………………………85 Chapter 4: Discussion and future directions……………………………….…………93 4.1 Discussion…………………………………………………………………………...93 4.2 Future directions…………………………………………………………………..108 4.2.1 Are schistosome Fox genes required for survival at each stage of developmental expression?..................................................................................108 4.2.2 Where is expression of Fox proteins localized in different developmental stages of the life cycle?........................................................................................109 3 4.2.3 What are the upstream regulators and downstream targets of SmFox genes?..................................................................................................109 Appendix: Immunolocalization of anti-Hsf1 to the acetabular glands of infectious schistosomes suggests a non-transcriptional function for this transcriptional activator…………………..……………………………………………………………111 5.1 Abstract…………………………………………………………………………….112 5.2 Author Summary………………………………………………………………….113 5.3 Introduction………………………………………………………………………..114 5.4 Materials and Methods……………………………………………………………117 5.4.1 Animals and parasites…………………………………………………….117 5.4.2 Preparation of schistosomal RNA………………………………………...117 5.4.3 Cloning……………………………………………………………………118 5.4.4 Yeast transformation and modified yeast one-hybrid…………………….118 5.4.5 Electrophoretic mobility shift assay (EMSA)…………………………….119 5.4.6 Comparison of protein sequences………………………………………...120 5.4.7 Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)…………………………………………………………….121 5.4.8 Recombinant protein purification………………………………………...122 5.4.9 Custom antibody production……………………………………………...122 5.4.10 Western blotting…………………………………………………………123 5.4.11 Immunohistochemistry………………………………………………….124 5.4.12 Imaging………………………………………………………………….125 4 5.5 Results……………………………………………………………………………...125 5.5.1 Schistosome Hsf1 is a transcriptional activator…………………………..125 5.5.2 SmHsf1 recognizes the heat shock DNA binding element from the schistosome HSP70 promoter…………………………………………………..128 5.5.3 SmHSF1 is expressed across schistosome developmental stages…….......132 5.5.4 A polyclonal antibody detects the SmHsf1 protein………………………133 5.5.5 Antibody raised against SmHSF1 localizes to the acetabular glands of S. mansoni cercariae………………………………………………….135 5.6 Discussion………………………………………………………………………….137 5.7 Acknowledgements………………………………………………………………..141 5.8 Supplementary Information……………………………………………………...142 Bibliography…………………………………………………………………………...144 5 List of Tables Table 2.1 Summary of blastp pairwise alignments of schistosome Fox protein DBDs (SmFoxA1-SmFoxG) and orthologs from mouse, fly, and roundworm…………………44 Table 2.2. Summary of SmFox yeast one-hybrid results………………………………..52 Supplementary Table 2.1. Databases and gene ID for forkhead homologs used in phylogenetic analysis…………………………………………………………………….55 Supplementary Table 2.2. Schistosome Forkhead gene ID numbers and oligonucleotide sequences used for Infusion cloning into the pGBKT7 vector…………………………..57 Supplementary Table 2.3. One-step RT-PCR reaction conditions used for cloning……58 Supplementary Table 2.4. Phusion PCR reaction conditions used for cloning…………59 Supplementary Table 2.5. Oligonucleotides used for absolute quantitative PCR analysis……………………………………………………………………………..59 Table 3.1. Summary of blastp pairwise alignments of schistosome Fox protein DBDs (SmFoxJ1-SmFoxP) and orthologs from mouse, fly, and roundworm…………………..71 Table 3.2. Summary of schistosome forkhead yeast one-hybrid results………………...81 Supplementary Table 3.1. Databases and accession numbers for forkhead homologs used in phylogenetic analysis………………………………………………………………….85 Supplementary Table 3.2. Gene ID numbers, plasmid names, and primers used for cloning schistosome forkhead genes……………………………………………………..88 6 Supplementary Table 3.3 Phusion PCR reaction conditions for cloning………………...90 Supplementary Table 3.4. One-step RT-PCR reaction conditions for cloning………….91 Supplementary Table 3.5. Primer sequences used for absolute quantitative PCR analysis……………………………………………………………………………..92 Table 4.1. Summary of SmFox yeast one-hybrid results………………………………106 Table S5.8.1. Names and sequences of oligonucleotides used for EMSA……………..142 7 List of Figures Figure 1.1. Global distribution of schistosome species infective to man………………..17 Figure 1.2. Life cycle of the schistosome worm, a parasitic trematode…………………21 Figure 1.3. The forkhead DNA binding domain of HNF-3 is similar in structure to histone H5………………………………………………………………….. 25 Figure 1.4. The insulin signaling pathway is conserved among a variety of organisms...28 Figure 2.1. Yeast expression vector pGBKT7 (Clontech, Mountain View, CA)……….34 Figure 2.2. Schematic of modified yeast one-hybrid……………………………………35 Figure 2.3 Schematic of SmFoxC isoforms……………………………………………...40 Figure 2.4. Phylogenetic analysis of schistosome Fox protein subclasses SmFoxA- SmFoxG………………………………………………………………………………….43 Figure 2.5. Schistosome forkhead gene expression varies during different stages of development……………………………………………………………………………...47 Figure 2.6. Schistosome forkhead transcription factors differ in ability to drive reporter gene expression in a yeast 1-hybrid analysis…………………………………………….50 Supplementary Figure 2.3.1. Yeast 1-hybrid analysis of SmFoxC isoforms…………….53 Supplementary Figure 2.3.2. X-Alpha-Galactosidase assay plates for yeast-1 hybrid analysis…………………………………………………………………………………...54 8 Figure 3.1 Schematic of SmFoxK2 isoforms…………………………………………….67 Figure 3.2 Phylogenetic analysis of schistosome Fox protein subclasses SmFoxJ- SmFoxP…………………………………………………………………………………..70 Figure 3.3. Schistosome forkhead genes are expressed across several stages of the life cycle……………………………………………………………………………………...74 Figures 3.4A and 3.4B. Several schistosome forkhead proteins are transcriptional activators…………………………………………………………………………………78 Supplementary Figure 3.3.1. Yeast 1-hybrid analysis
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